The field of cell reprogramming changed dramatically with the breakthrough publication by Yamanaka in 2006 in which he showed that mouse Embryonic Fibroblasts (MEFs) could be dedifferentiated into pluripotent stem cells through the forced retroviral mediated expression of four transcription factors (Oct 4, Sox 2, Klf 4, and c-Myc). With the promise of pluripotent cells while avoiding politically and ethically challenging Embryonic Stem (ES) Cells, the race was on to demonstrate the technique on human cells. Within one year, both Yamanakas and James Thompsons groups had demonstrated similar techniques with human fibroblasts. Whereas, Yamanaka used the same 4 factors, Thompson used Oct 4, Sox 2, Nanog and Lin 28 to reprogram the human somatic cells. The process of dedifferentiation of somatic cells was observed to involve an epigenetic based suppressed expression of the four exogenous transcription factors together with a lower level constitutive expression of the concomitant endogenous genes. Surprisingly, the failure of epigenetic suppression of the exogenous factors was already known to be associated with the failure of cellular reprogramming. However, despite the epigenetic suppression, the original mice generated by Yamanaka had a propensity to develop cancers associated with reactivation of the c-Myc gene. Furthermore, since genomic integration of viral LTR sequence can cause cellular transformation and Oct 4, and Nanog are sometimes elevated in cancers, it seemed unlikely that viral mediated iPS cells would ever be used for targeted stem cell mediated therapeutics. More recent work has shown that despite the continued suppression of the viral integrated exogenous transcription factors in iPS cells, the iPS cells remain less pluripotent than if the viral integrants are removed. The removal of these integrants has not proved possible to date, in any practical manner. For this reason, it would seem that the direct addition of the transcription factor proteins themselves to the cells (for a limited period of time) would be a perfect solution. The fly in the ointment here is the great difficulty in expressing these proteins in bioactive forms, especially Oct4, which is acknowledged as the one most critical factor. When expressed in E. coli., these transcription factors are sequestered in inclusion bodies as insoluble inactive proteins. Purification of these proteins requires denaturation in 8M urea which totally inactivates the protein and to date, renaturation has involved fast 20-fold dilution in urea free buffer. We have replicated this process and showed how this technique yields degraded and mostly inactive Oct4 and Sox2 protein. To remedy this, we have developed an automated process to refold these proteins expressed in E. coli., in a carefully controlled manner on solid support. We have used these proteins, renatured by this novel process to generate murine and human iPS colonies, which stain correctly but are not robust enough to serially passage. We attribute this to inadequate refolding fidelity of the Oct4 protein. We are continuing to refine the process while at the same time we are submitting the work to date for publication. In parallel experiments, we have succeeded in producing Oct4 and Sox2, in a Baculoviral based system, which, to our knowledge, has not been accomplished by any other group in the world to date. This protein, grown in insect SF-9 cells, is found exclusively in the nucleus of the cells expressing the protein, indicative of its solubility and bioactivity. Recently, we have purified this bioactive protein and identified it by Western blot. We are several weeks away from finishing the expression and purification of Nanog, in the same Baculoviral system. These three proteins, made as cell-membrane permeant proteins, expressed in SF-9 cells, should be adequate to generate robust murine and human iPS colonies when added to somatic cells intermittently for 8-12 days, based on our previous experiments. This technique can be adopted by researchers around the world to generate more pluripotent and reliable iPS cells for further research and perhaps therapy some day.